JP2020514368A - XVIII type collagen assay - Google Patents
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- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
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Abstract
本発明は、抗体に関し、その抗体は、短アイソフォームXVIII型コラーゲンと特異的に反応性であるが、中アイソフォームXVIII型コラーゲンと、または長アイソフォームXVIII型コラーゲンとは反応しない。本発明はまた、短アイソフォームXVIII型コラーゲンを検出または定量化するためのイムノアッセイの方法における抗体の使用に関し、方法は、血友病性疾患を評価するために使用され得る。The present invention relates to antibodies, which are specifically reactive with short isoform type XVIII collagen but not with medium isoform type XVIII collagen or with long isoform type XVIII collagen. The invention also relates to the use of the antibody in a method of an immunoassay for detecting or quantifying short isoform type XVIII collagen, which method can be used to assess hemophilia disease.
Description
本発明は、XVIII型コラーゲンの短アイソフォームを検出するための方法、および血友病性疾患の評価におけるその方法の使用に関する。 The present invention relates to a method for detecting short isoforms of collagen type XVIII, and the use of that method in the assessment of hemophilia disorders.
血管破裂による再発性関節血症は、血友病における主要な合併症であり、血友病性(HF)関節症を引き起こす進行性関節損傷の一因となっている。出血事象を低減するHF分野における医療ニーズは、血友病患者における年間出血率(ABR)の測定を必要とする。粗い尺度であるものの、ABRは、HF関節症と関連付けられる[1]が、臨床試験における重要なパラメータでもあり、患者に対する定量化可能な利益を保証する[2〜4]。内皮細胞機能障害およびマトリックス品質は、関節出血およびその後のHF関節症の発症と関連付けられ得る。 Recurrent arthremia due to vascular rupture is a major complication in hemophilia and contributes to the progressive joint damage that causes hemophilic (HF) arthropathy. The medical need in the field of HF to reduce bleeding events requires the measurement of annual bleeding rate (ABR) in hemophiliacs. Although being a crude measure, ABR is associated with HF arthropathy [1], but is also an important parameter in clinical trials and guarantees a quantifiable benefit to patients [2-4]. Endothelial cell dysfunction and matrix quality may be associated with joint bleeding and subsequent development of HF arthropathy.
血管破裂は、内皮細胞の直下に位置する基底膜(BM)の品質およびターンオーバーと関連付けられる。細胞外マトリックスターンオーバーは、上皮または内皮細胞損傷による多くの疾患における中心的な病理学的特徴である。内皮細胞機能は論じられているが、出血後、内皮細胞の基底をなすBMの露出をもたらす、血管内皮に対する損傷を特異的に定量化するための定量化可能な方法は存在しない。 Vascular rupture is associated with quality and turnover of the basement membrane (BM), which is located just below the endothelial cells. Extracellular matrix turnover is a central pathological feature in many diseases due to epithelial or endothelial cell damage. Although endothelial cell function has been discussed, there is no quantifiable method for specifically quantifying damage to the vascular endothelium that results in the exposure of the underlying BM of endothelial cells after bleeding.
内皮細胞に特異的なBMタンパク質を定量化することは、したがって、血友病患者における内皮細胞安定性および破裂に対する特定の関連性を有し得る。 Quantifying endothelial cell-specific BM proteins may therefore have particular relevance for endothelial cell stability and rupture in hemophiliacs.
IV、XV、およびXVIII型コラーゲンは、血管壁構造および膜の統合性の維持を担う、血管BMの最もよく知られているコラーゲンを表す(図1A)[5〜7]。 Collagen IV, XV, and XVIII represent the most well-known collagens in vascular BM (Fig. 1A) [5-7], which are responsible for maintaining the integrity of the vascular wall structure and membrane.
XVIII型コラーゲンは、様々な基底膜領域に局在する、短、中、および長の3つのアイソフォームで存在する[4〜6](図1B)。全ての3つのアイソフォームは、トロンボスポンジン1様ドメイン、および11個の非コラーゲンドメイン(NC1〜11)に隣接する10個の三重らせんコラーゲンドメイン(Col1〜10)を含有する。NC1ドメインは、抗血管新生特性を有するC末端エンドスタチンドメインを含有する[7]。短アイソフォームは、内皮特異的であり、血管中および筋肉構造の周りに見られる。ここには、ゼロまたは非常に低い量のみの中および長アイソフォームが存在する[5]。血管BMのリモデリング、損傷、および分解後、XVIII型コラーゲンの短アイソフォームは、他のタイプのコラーゲンで起こったように、影響および分解され、XVIII型コラーゲンの測定可能な断片を放出し得る[8、9]。 Collagen type XVIII exists in three short, medium, and long isoforms localized in various basement membrane regions [4-6] (FIG. 1B). All three isoforms contain a thrombospondin 1-like domain and 10 triple helix collagen domains (Col 1-10) flanked by 11 non-collagen domains (NC1-11). The NC1 domain contains a C-terminal endostatin domain with anti-angiogenic properties [7]. Short isoforms are endothelium-specific and are found in blood vessels and around muscle structures. There are only zero or very low amounts of medium and long isoforms [5]. After remodeling, damage, and degradation of vascular BM, the short isoforms of type XVIII collagen can be affected and degraded, as they did with other types of collagen, releasing measurable fragments of type XVIII collagen [. 8, 9].
XVIII型コラーゲンにおける突然変異はまた、常染色体劣性障害Knobloch症候群(KS)と関連付けられている。KSは、若齢での失明を引き起こす様々な眼球異常によって特徴付けられる[11、12]。また、col18a1−/−ノックアウトマウスは、網膜の表面に沿った硝子体における血管の退行遅延、網膜血管の血管新生機能障害、および虹彩BM構造変化を示した[8、13〜16]。よって、XVIII型コラーゲンは、目における血管形成を制御するために必須であり、場合によっては血管系全体のBM領域における重要な成分である[17]。 Mutations in collagen type XVIII have also been associated with the autosomal recessive disorder Knobloch syndrome (KS). KS is characterized by various ocular abnormalities that cause blindness at an early age [11,12]. Also, the col18a1 − / − knockout mice showed delayed regression of blood vessels in the vitreous along the surface of the retina, impaired neovascularization of retinal blood vessels, and iris BM structural change [8, 13-16]. Thus, collagen type XVIII is essential for controlling angiogenesis in the eye and is in some cases a key component in the BM region of the entire vasculature [17].
本出願人は、XVIII型コラーゲンの短アイソタイプに特異的ないずれのバイオマーカーおよび/または抗体についても把握していない。市販の抗体の大部分は、XVIII型コラーゲンのC末端エンドスタチン末端を認識するので、それらの抗体を用いて3つのアイソタイプを区別することはできない。 Applicants are not aware of any biomarkers and / or antibodies specific for the short isotype of type XVIII collagen. Since most of the commercially available antibodies recognize the C-terminal endostatin terminus of type XVIII collagen, these antibodies cannot be used to distinguish between the three isotypes.
よって、内皮XVIII型コラーゲン含有量の検出に関して血管特異的基底膜ターンオーバーを定量化するために、XVIII型コラーゲンの特異的短アイソタイプを測定し、2つの他のアイソフォームを除外する抗体および/またはバイオマーカーの必要性がある。 Thus, in order to quantify blood vessel-specific basement membrane turnover for the detection of endothelial type XVIII collagen content, a specific short isotype of type XVIII collagen was measured and an antibody and / or exclusion of two other isoforms was performed. There is a need for biomarkers.
出願人はここで、XVIII型コラーゲンの短アイソフォームを検出するためのアッセイを開発し、そのアッセイを使用して、HF関節症と診断された患者におけるXVIII型コラーゲンのターンオーバーの臨床的関連性を評価した。 Applicants have now developed an assay for the detection of short isoforms of type XVIII collagen, which is used to clinically relate turnover of type XVIII collagen in patients diagnosed with HF arthropathy. Was evaluated.
第1の態様では、本発明は、短アイソフォームXVIII型コラーゲンと特異的に反応性である抗体に関し、その抗体は、中アイソフォームXVIII型コラーゲンと、または長アイソフォームXVIII型コラーゲンとは反応しない。好ましくは、抗体は、短アイソフォームXVIII型コラーゲンのN末端エピトープと特異的に反応性である。好ましくは、N末端エピトープは、短アイソフォームXVIII型コラーゲンのN末端シグナルペプチドの切断および除去後に露出するエピトープである。好ましくは、抗体は、H2N−EPERISEEVG...(配列番号1)のN末端アミノ酸配列に含まれるN末端エピトープと特異的に反応性である。好ましくは、抗体は、N末端アミノ酸配列H2N−EPERIS...(配列番号2)を含むN末端エピトープと特異的に反応性である。 In a first aspect, the invention relates to an antibody that is specifically reactive with short isoform collagen XVIII, which antibody does not react with medium isoform collagen XVIII or with long isoform collagen XVIII. .. Preferably, the antibody is specifically reactive with the N-terminal epitope of short isoform type XVIII collagen. Preferably, the N-terminal epitope is the epitope exposed after cleavage and removal of the N-terminal signal peptide of short isoform type XVIII collagen. Preferably, the antibody, H 2 N-EPERISEEVG. . . It is specifically reactive with the N-terminal epitope contained in the N-terminal amino acid sequence of (SEQ ID NO: 1). Preferably, the antibody is N-terminal amino acid sequence H 2 N-EPERIS. . . It is specifically reactive with the N-terminal epitope containing (SEQ ID NO: 2).
好ましくは、抗体は、H2N−AEPERISEEVG(配列番号3)であるそのN末端アミノ酸配列のN拡張伸長バージョンを特異的に認識もしくは結合しない、かつ/またはH2N−PERISEEVG(配列番号4)であるそのN末端アミノ酸配列のN切断バージョンを特異的に認識もしくは結合しない。 Preferably, the antibody, H 2 N-AEPERISEEVG does not specifically recognize or bind the N extended extension version (SEQ ID NO: 3) in which the N-terminal amino acid sequence, and / or H 2 N-PERISEEVG (SEQ ID NO: 4) Does not specifically recognize or bind the N-truncated version of its N-terminal amino acid sequence.
抗体は、モノクローナルまたはポリクローナル抗体であり得る。好ましくは、抗体は、モノクローナル抗体である。 The antibody can be a monoclonal or polyclonal antibody. Preferably the antibody is a monoclonal antibody.
第2の態様では、本発明は、試料において短アイソフォームXVIII型コラーゲンを検出または定量化するためのイムノアッセイの方法に関し、その方法は、その短アイソフォームXVIII型コラーゲンを含む試料を上に記載の抗体と接触させることと、その抗体の結合の量を決定することと、を含む。 In a second aspect, the invention relates to a method of an immunoassay for detecting or quantifying short isoform type XVIII collagen in a sample, the method comprising a sample comprising the short isoform type XVIII collagen as described above. Contacting with the antibody and determining the amount of binding of the antibody.
本発明は、ヒト患者において短アイソフォームXVIII型コラーゲンを検出する方法を対象とし得、その方法は、
a.ヒト患者から試料を得ることと、
b.試料を抗体(上に記載)と接触させ、短アイソフォームXVIII型コラーゲンと抗体との間の結合を検出することによって、その短アイソフォームXVIII型コラーゲンが試料中に存在するかを検出することと、を含む。
The present invention may be directed to a method of detecting short isoform type XVIII collagen in a human patient, the method comprising:
a. Obtaining a sample from a human patient,
b. Detecting the presence of the short isoform XVIII collagen in the sample by contacting the sample with an antibody (described above) and detecting the binding between the short isoform collagen XVIII and the antibody. ,including.
好ましくは、方法は、短アイソフォームXVIII型コラーゲンのN末端エピトープを検出または定量化することを含む。N末端エピトープは、好ましくは、N末端アミノ酸配列H2N−EPERISEEVG...(配列番号1)に含まれる。好ましくは、N末端エピトープは、N末端アミノ酸配列H2N−EPERIS...(配列番号2)を含む。 Preferably, the method comprises detecting or quantifying the N-terminal epitope of short isoform collagen type XVIII. N-terminal epitope preferably, N-terminal amino acid sequence H 2 N-EPERISEEVG. . . (SEQ ID NO: 1). Preferably, N-terminal epitopes, N-terminal amino acid sequence H 2 N-EPERIS. . . (SEQ ID NO: 2) is included.
好ましくは、試料は、生物流体である。生物流体は、これらに限定されないが、血清、血漿、尿、脳脊髄液、または羊水であり得る。 Preferably the sample is a biofluid. The biofluid can be, but is not limited to, serum, plasma, urine, cerebrospinal fluid, or amniotic fluid.
イムノアッセイは、競合アッセイまたはサンドイッチアッセイであり得る。イムノアッセイは、ラジオイムノアッセイまたは酵素結合免疫吸着アッセイであり得る。 The immunoassay can be a competition assay or a sandwich assay. The immunoassay can be a radioimmunoassay or an enzyme-linked immunosorbent assay.
方法は、その方法によって決定されるその短アイソフォームXVIII型コラーゲンの量を、既知の疾患重症度の標準血友病性疾患試料と相関させて、血友病性疾患の重症度を評価することをさらに含み得る。これに関して、「標準血友病性疾患試料」は、既知の重症度の血友病性疾患を有することが知られる対象から得られる試料を意味する。 The method correlates the amount of its short isoform type XVIII collagen determined by the method with a standard hemophiliac disease sample of known disease severity to assess the severity of hemophiliac disease. Can be further included. In this regard, "standard hemophiliac disease sample" means a sample obtained from a subject known to have a hemophiliac disease of known severity.
あるいは、または加えて、方法は、その方法によって決定されるその短アイソフォームXVIII型コラーゲンの量を、健康な対象と関連した標準値と比較して、血友病性疾患の存在および/または重症度を評価することをさらに含み得る。これに関して、「健康な対象と関連した標準値」は、健康である、すなわち、血友病性疾患を有さないとみなされる対象について上に記載される方法によって決定される短アイソフォームXVIII型コラーゲンの標準化された量を意味する。標準化は、健康な対象の身長、体重、性別などに依存するであろう。 Alternatively, or in addition, the method compares the amount of its short isoform type XVIII collagen determined by the method with a standard value associated with a healthy subject to determine the presence and / or severity of a hemophilic disease. It may further include assessing the degree. In this regard, a "standard value associated with a healthy subject" is the short isoform Form XVIII as determined by the method described above for subjects who are considered to be healthy, ie, have no hemophiliac disease. Mean standardized amount of collagen. Standardization will depend on the height, weight, sex, etc. of a healthy subject.
あるいは、または加えて、方法は、対象から第1の時点および少なくとも1つのその後の時点で得られる少なくとも2つの試料中のXVIII型コラーゲンの量を定量化することをさらに含み得、第1の時点から少なくとも1つのその後の時点までのXVIII型コラーゲンの量の増加は、第1の時点から少なくとも1つのその後の時点までの血友病性疾患の悪化を示すか、または第1の時点から少なくとも1つのその後の時点までのXVIII型コラーゲンの量の減少は、第1の時点から少なくとも1つのその後の時点までの血友病性疾患の改善を示す。 Alternatively, or in addition, the method can further comprise quantifying the amount of type XVIII collagen in the at least two samples obtained from the subject at the first time point and at least one subsequent time point, the first time point. From at least one subsequent time point to an exacerbation of hemophilia from the first time point to at least one subsequent time point, or at least 1 time point from the first time point. A decrease in the amount of collagen type XVIII by one subsequent time point is indicative of an improvement in hemophilic disease from the first time point to at least one subsequent time point.
血友病性疾患は、血友病性関節症であり得る。上に記載される方法は、Knobloch症候群を評価するためにも使用され得る。 The hemophilic disease can be hemophilic arthropathy. The method described above can also be used to assess Knobloch syndrome.
別の態様では、本発明は、血友病性疾患を治療するための薬物の有効性を評価するための方法を対象とする。方法は、上に記載の方法を使用して、対象から第1の時点および対象への薬物の投与の期間中の少なくとも1つのその後の時点で得られる少なくとも2つの生物学的試料中のXVIII型コラーゲンの量を定量化することを含む。第1の時点から薬物の投与の期間中の少なくとも1つのその後の時点までのXVIII型コラーゲンの量の低減は、血友病性疾患を治療するための有効な薬物であることを示す。 In another aspect, the invention is directed to a method for assessing the efficacy of a drug for treating a hemophilic disease. The method uses Form XVIII in at least two biological samples obtained at a first time point from the subject and at least one subsequent time point during the period of administration of the drug to the subject using the method described above. Quantifying the amount of collagen. A reduction in the amount of type XVIII collagen from the first time point to at least one subsequent time point during the administration of the drug indicates that it is an effective drug for treating hemophilic disease.
最後の態様では、本発明は、短アイソフォームXVIII型コラーゲンの量を決定するためのアッセイキットに関し、上に記載の抗体と、
−ストレプトアビジン被覆96ウェルプレート
−その抗体と反応性であり、ビオチン化ペプチドH2N−EPERISEEVG−L−ビオチン(配列番号5)であり得、Lが、任意のリンカーである、ペプチド
−サンドイッチイムノアッセイにおける使用のための任意にビオチン化された二次抗体
−N末端配列H2N−EPERISEEVG...(配列番号6)を含むキャリブレータペプチド
−抗体HRP標識キット
−抗体放射性標識キット
−アッセイ可視化キットのうちの少なくとも1つと、を含む。
In a final aspect, the invention relates to an assay kit for determining the amount of short isoform type XVIII collagen, which comprises the antibody described above,
- streptavidin-coated 96-well plates - are reactive with the antibody, a biotinylated peptide H 2 N-EPERISEEVG-L- Biotin (SEQ ID NO: 5) to give, L is an optional linker, peptide - sandwich immunoassay optionally biotinylated secondary antibody -N-terminal sequence H 2 N-EPERISEEVG for use in. . . And at least one of a calibrator peptide containing (SEQ ID NO: 6), an antibody HRP labeling kit, an antibody radiolabeling kit, and an assay visualization kit.
定義
本明細書で使用されるとき、「N末端エピトープ」という用語は、ポリペプチドの末端にある、すなわち、ポリペプチドのN末端にある、N末端ペプチド配列を指し、その一般的な方向における意味として解釈されるものではない。
Definitions As used herein, the term "N-terminal epitope" refers to an N-terminal peptide sequence at the end of a polypeptide, ie, at the N-terminus of the polypeptide, and its meaning in its general orientation. Is not to be interpreted as.
本明細書で使用されるとき、「競合ELISA」という用語は、競合酵素結合免疫吸着アッセイを指し、当業者に知られている技法である。 As used herein, the term "competitive ELISA" refers to competitive enzyme-linked immunosorbent assay, a technique known to those of skill in the art.
本明細書で使用されるとき、「サンドイッチイムノアッセイ」という用語は、試料中の抗原の検出のための少なくとも2つの抗体の使用を指し、当業者に知られている技法である。 The term "sandwich immunoassay" as used herein refers to the use of at least two antibodies for the detection of antigen in a sample, a technique known to those of skill in the art.
本明細書で使用されるとき、「XVIII型コラーゲンの短アイソフォーム」という用語は、プロモーター1によって生成されるXVIII型コラーゲンのアイソフォームを指し、N末端非コラーゲン領域は、トロンボスポンジン−1様ドメイン(TSP−1)を含むが、未知機能のドメイン(DUF)またはFrizzledドメイン(FZ)を含有しない。「XVIII型コラーゲンの中アイソフォーム」という用語は、プロモーター2によって生成されるXVIII型コラーゲンの選択的スプライシングされたアイソフォームを指し、N末端非コラーゲン領域は、TSP−1およびDUFを含むが、FZを含まない。「XVIII型コラーゲンの長アイソフォーム」という用語は、プロモーター2によって生成されるXVIII型コラーゲンの選択的スプライシングされたアイソフォームを指し、N末端非コラーゲン領域は、TSP−1、DUF、およびFZを含む。 As used herein, the term "short isoform of collagen type XVIII" refers to the isoform of collagen type XVIII produced by promoter 1, wherein the N-terminal non-collagen region is a thrombospondin-1 like. It contains a domain (TSP-1) but no domain of unknown function (DUF) or Frizzled domain (FZ). The term "medium isoform of type XVIII collagen" refers to the alternatively spliced isoform of type XVIII collagen produced by promoter 2, where the N-terminal non-collagen region contains TSP-1 and DUF, but Does not include. The term "long isoform of type XVIII collagen" refers to the alternatively spliced isoform of type XVIII collagen produced by promoter 2, the N-terminal non-collagen region including TSP-1, DUF, and FZ. ..
本明細書で使用されるとき、「COL−18N」という用語は、XVIII型コラーゲンの短アイソフォームのN末端配列EPERISEEVG(配列番号1)に特異的な本明細書で開示されるアッセイを記載するための省略表現として使用される。 As used herein, the term "COL-18N" describes an assay disclosed herein that is specific for the N-terminal sequence EPERISEEVG (SEQ ID NO: 1) of the short isoform of type XVIII collagen. Used as a shorthand for.
本開示の実施形態は、以下の実施例に記載され、これは、本開示の理解を助けるために記述され、その後に続く特許請求の範囲において定義される本開示の範囲をいずれの方法でも限定するものと解釈されるべきではない。以下の例は、当業者に、記載される実施形態の作製および使用方法の完全な開示および説明を提供するように記述され、本開示の範囲を限定することは意図されず、以下の実験が行われた全てまたは唯一の実験であることを表すことも意図されない。使用される数値(例えば、量、温度など)に関する精度を確保する努力がなされたが、いくらかの実験誤差および偏差が考慮されるべきである。特に指示されない限り、部は重量部であり、分子量は重量平均分子量であり、温度は摂氏温度であり、圧力は大気またはそれに近い。 The embodiments of the present disclosure are described in the following examples, which in any way limit the scope of the present disclosure, which is described to aid the understanding of the present disclosure and is defined in the claims that follow. Should not be construed as doing. The following examples are described to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the described embodiments, and are not intended to limit the scope of the present disclosure, as the following experiments It is not intended to represent that all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (eg amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.
患者試料
血清を、35人の26歳以上の男性HF患者から収集した。このカットオフ年齢は、コラーゲンターンオーバーが約25歳の年齢での成長板の閉鎖で徐々に減少するため、選択された[21]。患者は、出血エピソード時の必要に応じた投薬、または5〜10IU/kgの組換えFVIII、2〜3回/週の低用量の予防薬の摂取のいずれかの治療歴を有した。患者は、世界血友病連盟身体検査スコア(Gilbertスコア)によって、およびPetterssonスコアに従った放射線学的評価によって定義される様々な程度のHF関節症を有した。患者の平均ABRは18.1であり、2〜46の範囲であった。除外基準は、血友病以外の出血障害、ヒト免疫不全ウイルス感染、慢性閉塞性肺疾患、関節疾患または肝線維症の病歴、および抗炎症性生物学的製剤またはステロイドでの治療であった。研究参加者は、Department of Haematology,Peking Union Medical College Hospital,Beijing,Chinaに登録された。研究は、Peking Union Medical College Hospital,Chinese Academy of Medical Sciences,Ethics Review Boardによって整理番号S−720で承認された。署名されたインフォームドコンセントを全ての対象から得た。
Patient Samples Serum was collected from 35 male HF patients over the age of 26. This cutoff age was chosen because collagen turnover gradually decreases with growth plate closure at the age of about 25 years [21]. Patients had a history of either medication as needed during the bleeding episode, or 5-10 IU / kg of recombinant FVIII, 2-3 times / wk low dose prophylactic drug. Patients had varying degrees of HF arthropathy as defined by the World Federation of Hemophilia Physical Examination Score (Gilbert score) and by radiological assessment according to the Petersson score. The patients average ABR was 18.1 and ranged from 2-46. Exclusion criteria were bleeding disorders other than hemophilia, human immunodeficiency virus infection, chronic obstructive pulmonary disease, history of joint disease or liver fibrosis, and treatment with anti-inflammatory biologics or steroids. Study participants were registered at Department of Haematology, Peking Union Medical College Hospital, Beijing, China. The study was approved by the Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Ethics Review Board, numbered S-720. Signed informed consent was obtained from all subjects.
COL−18Nのためのモノクローナル抗体開発
ヒトXVIII型コラーゲンα1鎖の短アイソフォームのN末端エピトープの最初の10個のアミノ酸に対応するペプチド(シグナルペプチド、34’EPERISEEVG’43を除く)を用いて、モノクローナルネオエピトープ特異的抗体を産生した。Beijing Administration Office of Laboratory AnimalおよびNordic Bioscienceの動物倫理委員会が動物実験を承認した。モノクローナル抗体の産生を、60μgのキーホールリンペットヘモシアニン(KLH)に結合したペプチドを含む200μLの乳化したフロイント完全アジュバントを用いる6〜8週齢Balb/Cマウスの皮下免疫化によって開始した。連続免疫化を、安定な力価レベルに到達するまで、フロイント不完全アジュバントにおいて2週間間隔で行った。マウスに100μLの0.9%塩化ナトリウム溶液中の50μgの免疫原を追加で静脈内投与し、3日後、脾臓細胞をSP2/0骨髄腫細胞(LGC Standards AB,Boras,Sweden)と融合した。ハイブリドーマを、96ウェルプレートにおいて成長させ、モノクローナル成長を、限界希釈によって確認した。クローンを、特異的エピトープ(EPERISEEVG、配列番号1)、伸長エピトープ(AEPERISEEVG、配列番号3)、および切断エピトープ(PERISEEVG、配列番号4)に対してスクリーニングした。mAb産生クローン、NB632−13H11/G5を、上記ペプチドに対する反応性に基づいて選択し、抗体を、Protein Gカラム(GE Healthcare,Hilleroed,Denmark)を用いて精製した。
The first 10 amino acids in the corresponding peptide of the short isoform of N-terminal epitope of the monoclonal antibody developed human type XVIII collagen α1 chain for COL-18N (signal peptide, 34 excluding 'EPERISEEVG' 43) with, A monoclonal neoepitope-specific antibody was produced. The Animal Administration Office of the Animal Administration Office of Laboratory Animal and the Animal Ethics Committee of Nordic Bioscience approved the animal experiments. Monoclonal antibody production was initiated by subcutaneous immunization of 6-8 week old Balb / C mice with 200 μL of emulsified Freund's complete adjuvant containing 60 μg of keyhole limpet hemocyanin (KLH) bound peptide. Serial immunizations were performed at 2 week intervals in Freund's incomplete adjuvant until stable titer levels were reached. Mice were additionally intravenously administered with 50 μg of immunogen in 100 μL of 0.9% sodium chloride solution, and 3 days later, spleen cells were fused with SP2 / 0 myeloma cells (LGC Standards AB, Boras, Sweden). Hybridomas were grown in 96 well plates and monoclonal growth was confirmed by limiting dilution. Clones were screened against a specific epitope (EPERISEEVG, SEQ ID NO: 1), an extended epitope (AEPERISEEVG, SEQ ID NO: 3), and a truncated epitope (PERISEEVG, SEQ ID NO: 4). The mAb producing clone, NB632-13H11 / G5, was selected based on its reactivity to the above peptides and the antibody was purified using a Protein G column (GE Healthcare, Hilleroed, Denmark).
COL−18N ELISAプロトコル
競合COL−18N ELISAを次の通り行った。96ウェルストレプトアビジン被覆プレート(Roche cat.:11940279)を、100μl/ウェルのコーティングバッファー(20mM Na2HPO4、3.7mM KH2PO4、137mM NaCl、2.7mM KCL、0.1%Tween20、1%BSA、pH7.4)に溶解した1.25ng/mLのビオチン化合成ペプチドEPERISEEVG−K−ビオチン(配列番号7)で被覆し、30分間20℃でインキュベートした。プレートを、洗浄バッファー(20mM Tris、50mM NaCl、pH7.2)で5回洗浄した。インキュベーションバッファー(20mM Na2HPO4、3.7mM KH2PO4、137mM NaCl、2.7mM KCL、0.1%Tween20、1%BSA、5%Liquid II pH7.4)に希釈した20μLの標準ペプチド(EPERISEEVG、配列番号6)または試料を、適切なウェルに添加し、続いて100μL/ウェルのモノクローナル抗体NB632−13H11/G5を添加し、1時間20℃でインキュベートした。洗浄後、100μlのウサギ抗マウス抗体(Jackson、315−035−045)を添加し、コーティングバッファーに1:3000で溶解し、1時間20℃、300rpmでインキュベートした。最後の5回の洗浄後、ウェルを100μLのテトラメチルベンジジン(TMB)(Kem−En−Tec cat.438OH)と暗所で15分間20℃、300rpmでインキュベートし、続いて100μL/ウェルの停止溶液(1%H2SO4)を添加した。比色反応を、650nmを参照として450nmで測定し、検量線を、4パラメトリック数理あてはめモデルを用いてプロットした。
COL-18N ELISA protocol Competitive COL-18N ELISA was performed as follows. 96-well streptavidin coated plates (Roche cat.:11940279) were coated with 100 μl / well of coating buffer (20 mM Na 2 HPO 4 , 3.7 mM KH 2 PO 4 , 137 mM NaCl, 2.7 mM KCL, 0.1% Tween 20, Coated with 1.25 ng / mL biotinylated synthetic peptide EPERISEVG-K-biotin (SEQ ID NO: 7) dissolved in 1% BSA, pH 7.4) and incubated for 30 minutes at 20 ° C. The plate was washed 5 times with wash buffer (20 mM Tris, 50 mM NaCl, pH 7.2). 20 μL of standard peptide diluted in incubation buffer (20 mM Na 2 HPO 4 , 3.7 mM KH 2 PO 4 , 137 mM NaCl, 2.7 mM KCL, 0.1% Tween 20, 1% BSA, 5% Liquid II pH 7.4). (EPERISEEVG, SEQ ID NO: 6) or sample was added to appropriate wells followed by 100 μL / well of monoclonal antibody NB632-13H11 / G5 and incubated for 1 hour at 20 ° C. After washing, 100 μl of rabbit anti-mouse antibody (Jackson, 315-035-045) was added, dissolved in coating buffer at 1: 3000, and incubated for 1 hour at 20 ° C. and 300 rpm. After the last 5 washes, the wells were incubated with 100 μL of tetramethylbenzidine (TMB) (Kem-En-Tec cat.438OH) in the dark for 15 minutes at 20 ° C., 300 rpm, followed by 100 μL / well of stop solution. (1% H 2 SO 4 ) was added. The colorimetric reaction was measured at 450 nm with 650 nm as reference and the calibration curve was plotted using a 4-parametric mathematical fit model.
COL−18N技術的評価
技術的アッセイ検証を国際的ガイドラインに従って行った。検出下限(LLOD)を、21個のゼロ試料(すなわち、アッセイバッファー)から決定される平均+3×標準偏差(SD)として計算した。検出上限(ULOD)を、標準A(1000ng/ml)の10個の測定値の平均−3×SDとして決定した。定量下限(LLOQ)を、30%未満の不正確性で可能な最低濃度によって決定した。アッセイ内およびアッセイ間変動を、2通りの10の独立したランによる7つのヒト試料の変動の平均として計算した。希釈回収を、2つのヒト血清および3つのヒトクエン酸血漿の2倍希釈物において決定し、未希釈物と比較した希釈マトリックスの回収パーセンテージとして計算した。添加回収を、測定範囲全体を網羅する濃度で標準ペプチドが添加されたヒト血清およびクエン酸血漿において、または濃度を2倍にするために同様の濃度の2つの試料を組み合わせることによって評価した。添加回収を、理論量の測定量回収パーセンテージとして計算した。ヘモグロビン、血清脂質、ビオチン、およびヒト抗マウス抗体(HAMA)によるマウス抗原に対するヒト抗体による干渉を、2倍希釈物を既知の濃度の血清試料に添加することによって決定した。濃度は、0.500mmol/lのヘモグロビン、0.56mmol/lの血清脂質、160μg/lのビオチン、および2010ng/mlのHAMAで開始した。回収パーセンテージを、正常血清試料を参照値として計算した。分析物安定性を、4つの凍結−融解サイクルにわたる2つの健康なヒト血清試料および1つの健康なクエン酸血漿試料について決定し、第1の凍結−融解サイクルの回収パーセンテージとして計算した。同じ試料を、非ストレス分析物に対して4℃および20℃で2時間、4時間、および24時間で試験した。最後に、抗体特異性を、標準(EPERISEEVG、配列番号1)、伸長(AEPERISEEVG、配列番号3)、切断(PERISEEVG、配列番号4)、および選択解除ペプチド(EPQIDEKKK(配列番号8)およびCPERALERR(配列番号9))に対する反応性を試験するサニティーチェックによって評価した。
COL-18N Technical Evaluation Technical assay validation was performed according to international guidelines. The lower limit of detection (LLOD) was calculated as the mean + 3 x standard deviation (SD) determined from 21 zero samples (ie assay buffer). The upper limit of detection (ULOD) was determined as the average of 10 measurements of standard A (1000 ng / ml) −3 × SD. The lower limit of quantification (LLOQ) was determined by the lowest concentration possible with an uncertainty of less than 30%. Intra-assay and inter-assay variability was calculated as the average of the variability of 7 human samples with 10 independent runs in duplicate. Dilution recovery was determined in 2-fold dilutions of 2 human sera and 3 human citrate plasma and calculated as the recovery percentage of the dilution matrix compared to undiluted. Spike recovery was assessed in human serum and citrate plasma spiked with standard peptide at concentrations covering the entire assay range, or by combining two samples of similar concentration to double the concentration. Spike recovery was calculated as the measured recovery rate of the theoretical amount. Human antibody interference against mouse antigens by hemoglobin, serum lipids, biotin, and human anti-mouse antibody (HAMA) was determined by adding a 2-fold dilution to a serum sample of known concentration. Concentrations started with 0.500 mmol / l hemoglobin, 0.56 mmol / l serum lipids, 160 μg / l biotin, and 2010 ng / ml HAMA. Percentage recovery was calculated using normal serum samples as reference values. Analyte stability was determined for two healthy human serum samples and one healthy citrate plasma sample over four freeze-thaw cycles and calculated as the recovery percentage of the first freeze-thaw cycle. The same samples were tested against unstressed analytes at 4 ° C and 20 ° C for 2 hours, 4 hours, and 24 hours. Finally, antibody specificity was determined by standard (EPERISEEVG, SEQ ID NO: 1), extension (AEPERISEEVG, SEQ ID NO: 3), cleavage (PERISEEVG, SEQ ID NO: 4), and deselection peptides (EPQIDEKKK (SEQ ID NO: 8) and CPERALERR (SEQ ID NO: 8). No. 9)) was evaluated by a sanity check that tests the reactivity.
統計
血清COL18−N濃度とABRとの間の相関は、GraphPad Prism v6(GraphPad Software,La Jolla,CA,USA)でSpearman順位相関係数を用いて分析した。差は、p<0.05である場合に統計的に有意とみなした。
Statistics The correlation between serum COL18-N concentration and ABR was analyzed with GraphPad Prism v6 (GraphPad Software, La Jolla, CA, USA) using the Spearman rank correlation coefficient. Differences were considered statistically significant when p <0.05.
結果および考察
ヒト血清および血漿(クエン酸、EDTA、ヘパリン)中のCOL−18Nを検出するためにモノクローナル抗体を用いる新規競合ELISAを開発および評価した。
Results and Discussion A new competitive ELISA using a monoclonal antibody to detect COL-18N in human serum and plasma (citrate, EDTA, heparin) was developed and evaluated.
主な所見は次の通りであった。
●血清COL−18Nレベルは、HF患者においてABRと相関した。
●許容可能なアッセイ内−アッセイ間変動ならびに許容可能な希釈および添加回収を有するヒト血清およびヒト血漿中のCOL−18Nを検出するための技術的に安定なアッセイ。
The main findings were as follows.
● Serum COL-18N levels correlated with ABR in HF patients.
A technically stable assay for detecting COL-18N in human serum and human plasma with acceptable intra-assay-interassay variability and acceptable dilution and spike recovery.
COL−18N ELISAの特徴分析
内皮BM分解を評価することができる競合COL−18N ELISAを開発した。ELISAの技術的性能を表1にまとめ、4.8〜671ng/mlの測定範囲、それぞれ7%および13%のアッセイ内およびアッセイ間変動性、100±20%以内の希釈および添加回収、ならびにイムノアッセイ干渉なしの分析安定性を提供する。血清(16.6ng/ml)、クエン酸血漿(12.5ng/ml)、EDTA血漿(13.2ng/ml)、およびヘパリン血漿(15.8ng/ml)中のCOL−18Nの正常濃度は、マトリックスにかかわらず一貫していた。
CHARACTERIZATION ANALYSIS OF COL-18N ELISA A competitive COL-18N ELISA was developed that can evaluate endothelial BM degradation. The technical performance of the ELISA is summarized in Table 1, measuring range of 4.8 to 671 ng / ml, intra and inter assay variability of 7% and 13% respectively, dilution and spike recovery within 100 ± 20%, and immunoassay. Provides analytical stability without interference. Normal concentrations of COL-18N in serum (16.6 ng / ml), citrate plasma (12.5 ng / ml), EDTA plasma (13.2 ng / ml), and heparin plasma (15.8 ng / ml) were: It was consistent regardless of the matrix.
NB632−13H11/G5抗体は、N末端XVIII型コラーゲンα1鎖の最初の10個のアミノ酸、短アイソフォーム(選択)を特異的に認識した(図2)。抗体は、関連ペプチドに対する反応性を示さないか、最小限の反応性を示し、高い特異性を示した(図2)。XVIII型コラーゲンの中および長形態のN末端との交差反応性の可能性は、もっともらしくない。XVIII型コラーゲンの3つのアイソフォームは、COL18A1遺伝子によって、2つの異なるプロモーターおよび選択的スプライシングの使用によってコードされる(18、19)(図1B)。結果として、短アイソフォームのN末端は、他の2つのアイソフォームのN末端とは全く異なってくる。 The NB632-13H11 / G5 antibody specifically recognized the first 10 amino acids, a short isoform (selection) of the N-terminal type XVIII collagen α1 chain (Fig. 2). The antibody showed no or minimal reactivity to the relevant peptides and showed high specificity (Fig. 2). The possibility of cross-reactivity with the medium and long forms of the N-terminus of type XVIII collagen is not plausible. The three isoforms of collagen type XVIII are encoded by the COL18A1 gene by the use of two different promoters and alternative splicing (18, 19) (FIG. 1B). As a result, the N-terminus of the short isoform is quite different from the N-terminus of the other two isoforms.
競合COL−18N ELISAの技術的評価は、アッセイの高い正確性および精度を含むXVIII型コラーゲンの血管形態のN末端に対する高い特異性を有する安定な感度のよいアッセイを明らかにした。
表1.COL−18N ELISAの技術的性能
Table 1. COL-18N ELISA technical performance
COL−18Nは、HF患者において年間出血率と相関する。出血性障害血友病は、適切な治療の不在下で不可避の関節症を引き起こす反復性関節血症によって臨床的に明らかになる。血友病における医薬介入の1つの基本的な特徴は、ABRの低下であるが、高分解能を有する客観的な定量化可能なパラメータが欠如している。 COL-18N correlates with annual bleeding rate in HF patients. Hemorrhagic haemophilia is clinically manifested by recurrent arthremia that causes unavoidable arthropathy in the absence of appropriate treatment. One fundamental feature of medicinal interventions in hemophilia is a reduction in ABR, but a lack of objective quantifiable parameters with high resolution.
血友病の出血重症度は、一般的には血漿中のFVIII/IX活性の程度と反比例するが、出血傾向の実質的な変動性は周知である。低減した特発性出血およびより低い因子濃縮物の要件が、重症HF患者の10〜15%のサブセットで報告されている[23、24]。また、非重症HF患者における阻害剤の開発は、出血表現型をかなり高め得る[25]。 Although hemorrhagic bleeding severity is generally inversely proportional to the extent of FVIII / IX activity in plasma, the substantial variability of bleeding tendency is well known. Reduced idiopathic bleeding and lower factor concentrate requirements have been reported in a subset of 10-15% of patients with severe HF [23,24]. Also, the development of inhibitors in non-severe HF patients can significantly enhance the hemorrhagic phenotype [25].
出血表現型は、アッセイの標準化によって引き起こされるFVIIIアッセイ間の大きな相違によってさらに損なわれ得[26]、療法中に使用されるFVIII濃縮物のタイプによっても影響され得る[27、28]。他のアッセイは、トロンビン生成と同様に、HF患者において出血表現型と相関する[29]が、トロンビン生成の発生にかかわらずFVIII阻害剤を有するHF患者において一貫性がない[30]。 The hemorrhagic phenotype can be further compromised by the large differences between FVIII assays caused by assay standardization [26] and can also be affected by the type of FVIII concentrate used during therapy [27, 28]. Other assays, like thrombin generation, correlate with a hemorrhagic phenotype in HF patients [29], but are inconsistent in HF patients with FVIII inhibitors despite the occurrence of thrombin generation [30].
血友病では、i)内皮細胞損傷、ii)出血、ならびにiii)血栓形成および創傷治癒の遅延の結果として、血友病の臨床症状および病態生理学的疾患表現の一因となる内皮リモデリングが影響を受け得る。本明細書に記載されるアッセイを使用して、血管内皮XVIII型コラーゲンは、HF患者においてABRと相関することがここで見出された(図3、r=0.45、p<0.006)。病理学的プロセスの客観的バイオマーカーは、ABRと関連する分解されたXVIII型コラーゲンのものと同様に、治療をベンチマークし、患者を監視するのを補助し得、結果として患者の利益のための薬物開発を補助し得る。 In hemophilia, i) endothelial cell damage, ii) bleeding, and iii) delayed thrombus formation and wound healing result in endothelial remodeling that contributes to the clinical manifestations and pathophysiological disease manifestations of hemophilia. Can be affected. Using the assay described herein, vascular endothelial type XVIII collagen was found here to correlate with ABR in HF patients (FIG. 3, r = 0.45, p <0.006). ). Objective biomarkers of pathological processes, like those of degraded type XVIII collagen associated with ABR, may help benchmark treatment and monitor patients, resulting in benefit to the patient. Can assist drug development.
結論
要約すると、組み合わされたデータは、技術的にロバストなCOL−18Nバイオマーカーが、XVIII型コラーゲンの短アイソフォームの分解に影響を及ぼす、血管BM分解およびリモデリングを含む病理に関連し得ることを示す。加えて、データは、COL−18Nバイオマーカーが、関節症の発症を予防するために患者の最適な治療および監視のためにABRを評価することを可能にする。
CONCLUSIONS In summary, the combined data suggest that technically robust COL-18N biomarkers may be associated with pathologies involving vascular BM degradation and remodeling that affect the degradation of short isoforms of type XVIII collagen. Indicates. In addition, the data allow the COL-18N biomarker to assess ABR for optimal treatment and monitoring of patients to prevent the development of arthropathy.
本明細書において、特に明示的に指示されない限り、「または(論理和)」という語は、条件のうちの1つのみが満たされることを必要とする演算子「排他的論理和」とは対照的に、記述される条件の一方または両方が満たされる場合に真の値を返すという演算子の意味で使用される。「含む(comprising)」という語は、「からなる(consisting of)」を意味するものではなく、「含む(including)」の意味で使用される。上記で認識される全ての先行する教示は、本明細書に参照により組み込まれる。いずれの先行公表文献の認識も、その教示が、これに関する日付でオーストラリアまたは他の場所において共有一般知識であったことを認めるか、または表すものであると解釈されるべきではない。
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17 Aikio M,Elamaa H,Vicente D,Izzi V,Kaur I,Seppinen L,Speedy HE,Kaminska D,Kuusisto S,Sormunen R,Heljasvaara R,Jones EL,Muilu M,Jauhiainen M,Pihlajamaki J,Savolainen MJ,Shoulders CC,Pihlajaniemi T.Specific collagen XVIII isoforms promote adipose tissue accrual via mechanisms determining adipocyte number and affect fat deposition.Proc Natl Acad Sci U S A 2014;111:E3043−52.
18 Carmeliet P,Collen D.Molecular Analysis of Blood Vessel Formation and Disease.Am J Physiol 1997;273:2091−104.
19 Karsdal MA,Bay−Jensen AC,Leeming DJ,Henriksen K,Christiansen C.Quantification of “end products” of tissue destruction in inflammation may reflect convergence of cytokine and signaling pathways − implications for modern clinical chemistry.Biomarkers 2013;18:375−8.
20 Karsdal MA,Nielsen MJ,Sand JM,Henriksen K,Genovese F,Bay−Jensen A−C,Smith V,Adamkewicz JI,Christiansen C,Leeming DJ.Extracellular Matrix Remodeling:The Common Denominator in Connective Tissue Diseases Possibilities for Evaluation and Current Understanding of the Matrix as More Than a Passive Architecture,but a Key Player in Tissue Failure.Assay Drug Dev Technol 2013;11:70−92.
21 Iki M,Akiba T,Matsumoto T,Nishino H,Kagamimori S,Kagawa Y,Yoneshima H.Reference database of biochemical markers of bone turnover for the Japanese female population.Japanese Population−based Osteoporosis(JPOS)Study.Osteoporos Int 2004;15:981−91.
22 Gefter ML,Margulies DH,Scharff MD.A simple method for polyethylene glycol−promoted hybridization of mouse myeloma cells.Somatic Cell Genet 1977;3:231−6.
23 Aledort LM,Haschmeyer RH,Pettersson H.A longitudinal study of orthopaedic outcomes for severe factor−VIII−deficient haemophiliacs.The Orthopaedic Outcome Study Group.J Intern Med 1994;236:391−9.
24 Aznar JA,Magallon M,Querol F,Gorina E,Tusell JM.The orthopaedic status of severe haemophiliacs in Spain.Haemophilia 2000;6:170−6.
25 van Velzen AS,Eckhardt CL,Streefkerk N,Peters M,Hart DP,Hamulyak K,Klamroth R,Meijer K,Nijziel M,Schinco P,Yee TT,van der Bom JG,Fijnvandraat K.The incidence and treatment of bleeding episodes in non−severe haemophilia A patients with inhibitors.Thromb Haemost 2015;115:543−50.
26 Barrowcliffe TW,Raut S,Sands D,Hubbard AR.Coagulation and Chromogenic Assays of Factor VIII Activity:General Aspects,Standardization,and Recommendations.Semin Thromb Hemost 2002;28:247−56.
27 Mikaelsson M.Influence of phospholipids on the assessment of factor VIII activity.Haemophilia 1998;4.
28 Mikaelsson M,Oswaldsson U.Assaying the Circulating Factor VIII Activity in Hemophilia A Patients Treated with Recombinant Factor VIII Products.Semin Thromb Hemost 2002;28:257−64.
29 Brummel−Ziedins KE,Branda RF,Butenas S,Mann KG.Discordant fibrin formation in hemophilia.J Thromb Haemost 2009;7:825−32.
30 Ragni M V.,DiMichele DM,Hay CM,Malec LM,Seaman CD,Li J,Yabes JG,Butenas S,Brummel−Ziedins K.Thrombin generation and bleeding in haemophilia inhibitor patients during immune tolerance induction.Haemophilia 2016;22:240−7.
In this specification, unless stated otherwise explicitly, the term "or (or)" is in contrast to the operator "exclusive or" which requires that only one of the conditions is met. Specifically, it is used in the sense of an operator that returns a true value if one or both of the described conditions are met. The word "comprising" is used in the sense of "including" rather than in "consisting of." All preceding teachings recognized above are incorporated herein by reference. The recognition of any prior publication should not be construed as an admission or representation that the teachings were common general knowledge in Australia or elsewhere as of a date related thereto.
Reference 1 Funk MB, Schmidt H, Becker S, Escuriola C, Klarmann D, Klingebieel T, Kreuz W. Modified magnetic resonance imaging score compared with orthopedic and radiological scores for the evaluation of haemophilic arthropathy. Haemophilia 2002; 8: 98-103.
2 Fischer K, Steen Carlsson K, Petrini P, Holmstrom M, Ljung R, van den Berg HM, Berntorp E. et al. Intermediate-dose versus high-dose profilaxis for save hemophilia: comparing outcome and costs since the 1970s. Blood 2013; 122: 1129-36.
3 Gringeria, Lundin B, von Mackensen S, Mantovani L, Mannucci PM. A randomized clinical trial of profilaxis in children with hemophilia A (the ESPRIT Study). J Thromb Haemost 2011; 9: 700-10.
4 Lundin B, Ljung R, Pettersson H .; MRI scores of ankle joints in children with haemophilia-comparison with clinical data. Haemophilia 2005; 11: 116-22.
5 Tomono Y, Naito I, Ando K, Yonezawa T, Sado Y, Hirakawa S, Arata J, Okigaki T, Ninomiya Y. Epitope-defined monoclonal antioxidants against multiplexin collagens demonstrate that type XV and XVIII collages are exposed compressed. Cell Struct Funct 2002; 27: 9-20.
6 Ricard-Blum S, Ruggiero F. The collagen superfamily: from the extracellular matrix to the cell membrane. Pathol Biol 2005; 53: 430-42.
7 Manon-Jensen T, Kjeld NG, Karsdal MA. Collagen-mediated hemostasis. J Thromb Haemost 2016; 14: 438-48.
8 Elamaa H, Snellman A, Rehn M, Audio-Harmainen H, Pihlajaniemi T .; Characterization of the human type XVIII collagen gene and proteolytic processing and sustenance of the variant contended. Matrix Biol 2003; 22: 427-42.
9 Saarela J, Rehn M, Oikarinen a, Audio-Harmainen H, Pihlajaniemi T. The short and long forms of type XVIII collagen shoW clear clear suspecti? Cients in location membranes membranes. Am J Pathomeric American Society for Investigative Pathology; 1998; 153: 611-26.
10 Seppinen L, Pihlajaniemi T. The multiple functions of collagen XVIII in development and disease. Matrix Biol International Society of Matrix Biology; 2011; 30: 83-92.
11 Marneros AG, Olsen BR. Physiologic role of collagen XVIII and endostatin. FASEB J 2005; 19: 716-28.
12 Passos-Bueno MR, Suzuki OT, Armelin-Correa LM, Sertie AL, Errera FIV, Bagatini K, Kok F, Leite KRM. Mutations in collagen 18A1 (COL18A1) and ther relevance to the human phenotype. An Acad Bras Science 2006; 78: 123-31.
13 Marneros AG, Keene DR, Hansen U, Fukai N, Molton K, Goletz PL, Moiseyev G, Pawlyk BS, Halfter W, Dong S, Shibata M, Li T, Bruch Ruk. Collagen XVIII / endostatin is essential for vision and retinal pigment epithelial function. EMBO J 2004; 23: 89-99.
14 Utriainen A, Eklund L, Pihlajaniemi and T.P. Structurally altered basement membranes and hydrocephalus in a type XVIII collagen defensive mouse line. Hum Mol Genet 2004; 13: 2089-99.
15 Marneros AG, Olsen BR. Age-Dependent Iris Abnormalities in Collagen XVIII / Endostatin Deficient Mice with Human Pigment Dispersion Syndrome. Investigation Ophthalmology Vis Sci 2003; 44: 2367.
16 Fukai N.V. Lack of collagen XVIII / endostatin results in eye abnormalities. EMBO J 2002; 21: 1535-44.
17 Aikio M, Elama H, Vicente D, Izzi V, Kaur I, Seppinen L, Speedy HE, Kaminska D, Kuusto S, Sorjuna la Jalu, Jluas, Jelusvaara R, Jonaslava R, Jonva, R, Jelusvaara R, Jonaslava R, Jonas el. CC, Pihlajaniemi T. Specific Collagen XVIII isoforms promote adipose tissue accu ial via mechanisms deter ing adipose number and affect fat deposition. Proc Natl Acad Sci USA 2014; 111: E3043-52.
18 Carmeliet P, Collen D. et al. Molecular Analysis of Blood Vessel Formation and Disease. Am J Physiol 1997; 273: 2091-104.
19 Karsdal MA, Bay-Jensen AC, Leeming DJ, Henriksen K, Christiansen C .; Quantification of “end products” of tissu destruction in flamm ay reflex connec tion and reconciliation and signature pacification. Biomarkers 2013; 18: 375-8.
20 Karsdal MA, Nielsen MJ, Sand JM, Henriksen K, Genovese F, Bay-Jensen A-C, Smith V, Adamkewicz JI, Christians C, Leeming DJ. Extracellular Matrix Remodeling: The Common Denominator in Connective Tissue Diseases Possibilities for Evaluation and Current Understanding of the Matrix as More Than a Passive Architecture, but a Key Player in Tissue Failure. Assay Drug Dev Technol 2013; 11: 70-92.
21 Iki M, Akiba T, Matsumoto T, Nishino H, Kagamimori S, Kagawa Y, Yoneshima H .; Reference database of biochemical markers of bone turnover for the Japan female population. Japan Population-based Osteoporosis (JPOS) Study. Osteoporos Int 2004; 15: 981-91.
22 Gefter ML, Margulies DH, Scharff MD. A simple method for polyethylene glycol-promoted hybridization of mouse myeloma cells. Somatic Cell Genet 1977; 3: 231-6.
23 Aledort LM, Haschmeyer RH, Pettersson H .; A long-term study of orthopaedic outcomes for save factor-VIII-definient haemophiliacs. The Orthopedic Outcome Study Group. J Intern Med 1994; 236: 391-9.
24 Aznar JA, Magallon M, Querol F, Gorina E, Tusell JM. The orthopaedic status of save haemophiliacs in Spain. Haemophilia 2000; 6: 170-6.
25 van Velzen AS, Eckhardt CL, Strefkerk N, Peters M, Hart DP, Hamulyak K, Klamroth R, Meijer K, Nijziel M, Schinco P, Yeat Jr. The incidence and treatment of bleeding episodes in non-sever haemophilia A patents with inhibitors. Thromb Haemost 2015; 115: 543-50.
26 Barroughcliffe TW, Raut S, Sands D, Hubbard AR. Coagulation and Chromogenic Assays of Factor VIII Activity: General Aspects, Standardization, and Recommendations. Semin Thromb Hemost 2002; 28: 247-56.
27 Mikaelsson M .; Influence of phospholipids on the assessment of factor VIII activity. Haemophilia 1998; 4.
28 Mikaelsson M, Oswaldsson U .; Assaying the Circulating Factor VIII Activity in Hemophilia A Patients Treated with Recombinant Factor VIII Products. Semin Thromb Hemost 2002; 28: 257-64.
29 Brummel-Ziedins KE, Branda RF, Butenas S, Mann KG. Discordant fibrin formation in hemophilia. J Thromb Haemost 2009; 7: 825-32.
30 Ragnei MV. , DiMichel DM, Hay CM, Malec LM, Seaman CD, Li J, Yabes JG, Butenas S, Brummel-Ziedins K .; Thrombin generation and bleeding in haemophilia inhibitor patents duration immunity tolerance induction. Haemophilia 2016; 22: 240-7.
Claims (20)
前記第1の時点から前記少なくとも1つのその後の時点までのXVIII型コラーゲンの量の増加が、前記第1の時点から前記少なくとも1つのその後の時点までの血友病性疾患の悪化を示し、
前記第1の時点から前記少なくとも1つのその後の時点までのXVIII型コラーゲンの量の減少が、前記第1の時点から前記少なくとも1つのその後の時点までの血友病性疾患の改善を示す、請求項7〜14のいずれか一項に記載の方法。 The method further comprises quantifying the amount of type XVIII collagen in at least two samples obtained from the subject at a first time point and at least one subsequent time point,
An increase in the amount of type XVIII collagen from the first time point to the at least one subsequent time point indicates an exacerbation of hemophiliac disease from the first time point to the at least one subsequent time point,
A reduction in the amount of type XVIII collagen from the first time point to the at least one subsequent time point indicates an improvement in hemophilic disease from the first time point to the at least one subsequent time point. Item 15. The method according to any one of items 7 to 14.
前記第1の時点から前記薬物の投与の期間中の前記少なくとも1つのその後の時点までのXVIII型コラーゲンの量の低減が、血友病性疾患を治療するための有効な薬物であることを示す、方法。 A method for assessing the efficacy of a drug for treating a hemophilic disease, said method comprising: using a method according to any one of claims 7-14 from a subject. Quantifying the amount of type XVIII collagen in at least two samples obtained at one time point and at least one subsequent time point during the administration of the drug to the subject,
Reducing the amount of collagen type XVIII from said first time point to said at least one subsequent time point during the administration of said drug is an effective drug for treating hemophilia disease ,Method.
−ストレプトアビジン被覆96ウェルプレート
−前記抗体と反応性であり、ビオチン化ペプチドH2N−EPERISEEVG−L−ビオチン(配列番号5)であり得、Lが、任意のリンカーである、ペプチド
−サンドイッチイムノアッセイにおける使用のための任意にビオチン化された二次抗体
−N末端配列H2N−EPERISEEVG・・・(配列番号6)を含むキャリブレータペプチド
−抗体HRP標識キット
−抗体放射性標識キット
−アッセイ可視化キットのうちの少なくとも1つと、を含む、キット。 An assay kit for determining the amount of short isoform type XVIII collagen, wherein the kit comprises the antibody according to any one of claims 1 to 6,
- streptavidin-coated 96-well plates - are reactive with the antibody, a biotinylated peptide H 2 N-EPERISEEVG-L- Biotin (SEQ ID NO: 5) to give, L is an optional linker, peptide - sandwich immunoassay optionally biotinylated secondary antibody -N-terminal sequence H 2 N-EPERISEEVG ··· (SEQ ID NO: 6) calibrator peptide including for use in - assay visualization kit - antibody HRP labeled kit - antibody radiolabeling kits A kit comprising at least one of and.
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AM. J. PATHOL., vol. 153, JPN6021051639, 1998, pages 611 - 626, ISSN: 0004881572 * |
J. THROMB. HAEMOST., vol. 14, JPN6021051635, 2016, pages 438 - 448, ISSN: 0004881576 * |
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MATRIX BIOL., vol. 22, JPN6021051636, 2003, pages 427 - 442, ISSN: 0004881575 * |
MATRIX BIOL., vol. 30, JPN6021051638, 2011, pages 83 - 92, ISSN: 0004881573 * |
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